In cAMP-dependent protein kinase (PKA) two highly conserved signaling mechanisms have converged. The catalytic (C) subunit of PKA serves as the prototype for the protein kinase superfamily, while the regulatory (R) subunits are the major receptors for cAMP. cAMP and the cyclic nucleotide binding (CNB) domain have co- evolved from bacteria to man as a mechanism to translate an extra-cellular stress signal into a biological response. The long term goals for this grant have been to elucidate the structure and function of the PKA regulatory subunits and the corresponding holoenzymes. At the beginning of the past granting period we had just solved our first holoenzyme complex of the C- subunit bound to a deletion mutant of RI1 that contained a single CNB domain. Subsequent structures of RI1 and RII1 heterodimers containing both CNB domains allowed us to understand inhibition and allosteric activation by cAMP. With holoenzyme complexes of RI1, RII1, and RII2 now solved, we now are poised to understand how the highly complex and dynamic tetrameric holoenzymes are assembled in novel ways and how they are allosterically activated by cAMP. The tetramers are the physiologically relevant forms of PKA that are targeted to specific sites in the cell. Furthermore, based on small angle scattering, the global architecture of RI1, RII1 and RII2 holoenzymes are very different. These isoforms are functionally non-redundant and contribute enormously to the specificity of PKA signaling. Our goals over the next granting period are to elucidate the structure and the molecular organization of the tetrameric PKA holoenzymes. Specific Aim I is to elucidate the structure of the RII2 tetrameric holoenzyme. Understanding the organization of the RI1 tetrameric holoenzyme is the focus of Specific Aim II. Specific Aim III is aimed at elucidating the dynamic features of the linker regions of the PKA regulatory subunits as they toggle between the holoenzyme state and the cAMP bound state. These linker regions define the different architecture of the RI and RII holoenzymes. Activation of each holoenzyme is highly allosteric but the detailed mechanisms are different. These isoform differences are crucial for PKA signaling at localized sites in the cell. Specific Aim IV is exploratory and lays the foundation for future studies, which will require cryo electron microscopy to study PKA as part of a larger signaling complex. Achieving these goals demands a highly interdisciplinary strategy. We will therefore use solution methods such as small angle Xray scattering, small angle neutron scattering, stopped-flow fluorescence, fluorescence resonance energy transfer, hydrogen/deuterium exchange coupled with mass spectrometry, and NMR spectroscopy in parallel with Xray crystallography. We have established a very effective collaborative team to explore comprehensively at high resolution and in solution the structure, function and dynamics of PKA signaling. PUBLIC HEALTH RELEVANCE: Understanding how cells convert an extracellular signal into a biological response is central to all of biology. With PKA, a ubiquitous cAMP-dependent protein kinase that regulates processes as diverse as memory and development, we have the potential to unravel fundamental signaling responses in great molecular detail. The work will not only enhance our basic understanding of these molecular switches but will also open the door for the design of new therapeutics capable of interfering with cell signaling.